THE HCMV INTERACTOME: A QUANTITATIVE ANALYSIS OF HUMAN CYTOMEGALOVIRUS-HOST PROTEIN INTERACTIONS Luís Miguel Veiga Nobre Department of Medicine University of Cambridge Darwin College January 2020 This dissertation is submitted for the degree of Doctor of Philosophy THE HCMV INTERACTOME: A QUANTITATIVE ANALYSIS OF HUMAN CYTOMEGALOVIRUS-HOST PROTEIN INTERACTIONS Luís Miguel Veiga Nobre Summary Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the adult population. Similarly to other herpesvirus, it is able to enter a state of latency, persisting within the host for life. Infection of healthy individuals is normally asymptomatic, however it is among the most common causes of allograft rejection, and can lead to several life-threatening diseases in the immunocompromised. Moreover, it is the leading viral infectious cause for congenital disease. HCMV encodes 171 canonical genes, and a substantial number of non-canonical ORFs have been identified by ribosome profiling and proteomics. The functions of many canonical HCMV proteins remain poorly understood, and it is not yet clear how many non-canonical ORFs encode functional polypeptides. Recent studies have provided an extensive overview on the modulation of gene expression as well as the spatio-temporal dynamics of viral and host proteins during HCMV infection. However, characterisation of specific protein-protein interactions and the exact molecular mechanisms underpinning the biological changes observed during viral infection are beyond the scope of these approaches. Affinity-purification mass spectrometry was performed to identify binding partners for 169 canonical, and 2 non-canonical HCMV proteins in infected cells. CompPass filtering determined an extensive network of high-confidence interacting proteins, with >3,400 virus-host and >150 virus-virus protein interactions. I Domain association analysis identified protein domains co-occurring with unusual frequency, while functional enrichment analysis provided an insight into novel functions of multiple viral genes as well as how HCMV systematically modulates host environment, for example interacting with transcriptional repressive complexes or families of ubiquitin E3 ligases. Furthermore, combining interaction data with a recently published systematic analysis of HCMV-induced protein degradation identified viral interactors for 31/133 degraded host targets. Finally, the uncharacterised, non-canonical ORFL147C protein was found to interact with elements of the mRNA splicing machinery, and a mutational study suggested its importance in viral replication. The interactome data will be important for future studies of herpesvirus infection. II Declaration I hereby declare, that except where specific reference is made to the work of others, the contents of this dissertation are original and have not been submitted in whole or in part for consideration for any other degree of qualification in this, or any other university. This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as where specified in the text and Acknowledgments. This dissertation does not exceed the specified word limit of 60,000 words as defined by the Degree Committee, excluding figures, photographs, tables, appendices and bibliography. Luís Miguel Veiga Nobre January 2020 III Acknowledgments First and foremost, I would like to thank my supervisor Dr. Mike Weekes for guidance, encouragement, and continued support throughout this project. His mentorship has truly been crucial for my development as a research scientist and I am very grateful for the opportunities and teaching he has provided. I am also grateful for the fantastic work environment provided by members of the Weekes lab, with special thanks to Dr. Katie Nightingale for helpful discussions, willingness to help with experiments and interpretation of results; and Miss Lior Soday for the R scripts that facilitated disentanglement of peptide assignment between canonical and non-canonical ORFs. I am also extremely grateful to the Wellcome Trust for funding this project. I would like to thank Dr. Robin Antrobus, from the CIMR Proteomics Core facility, for his diligence and heedfulness in handling the multitude of mass spectrometry samples required for this project. I am very grateful for the invaluable help provided by Dr. Edward Huttlin, from the immunoprecipitation protocol, to bioinformatics tools to filter the dataset and identify protein domain associations, as well as general advice on the project and data handling. I would like to express deep gratitude to Professor Steven Gygi and his lab, for access to the mass spectrometry data processing platform which has been fundamental to this project and many other collaborative works that I have been involved with. I would also like to thank our several collaborators from Cardiff University: Dr. Rich Stanton, Dr. Pete Tomasec, Dr. Eddie Wang and Professor Gavin Wilkinson, for stimulating discussions and generation of viral resources; Dr. James Davies and Dr. Sepehr Seirafian for generating the recombinant adenovirus library which provided a large proportion of templates for the cloning of HCMV genes into the pHAGE-SFFV library. My family for granting me the opportunities that have led me here, and my closest friends for the relentless support and patience they’ve shown in the past four years. Finally, I would like to dedicate this work to the loving memory of Mr. Aleix Gorchs Rovira, who accompanied me throughout this journey. Aleix wrote up his PhD thesis alongside me but passed away before submitting a finalised dissertation. IV Table of Contents List of Figures 5 List of Tables 8 List of Abbreviations 9 1 | Introduction 12 1.1 Herpesviridae 12 1.1.1 Human Herpesvirus 15 1.2 Human cytomegalovirus 16 1.2.1 Virion structure 16 1.2.1.1 Capsid 17 1.2.1.2 Tegument 19 1.2.1.3 Envelope 21 1.2.1.4 Other virion components 22 1.2.1.5 Non-infectious viral particles 23 1.2.2 HCMV genome 23 1.2.2.1 Cis-acting sequences 24 1.2.2.2 Gene products 25 1.2.2.3 Viral DNA replication 29 1.2.3 Lytic lifecycle 30 1.2.4 Viral latency 33 1.2.5 Transmission and tropism 33 1.2.6 Strains of HCMV 34 1.2.7 Immune response and modulation 36 1.2.7.1 Innate immune response 36 1.2.7.2 Adaptive immune response 37 1.2.7.3 Immune evasion 37 1.2.8 Antiviral treatment 39 1.3 Proteomics as a tool for studying viral infection 40 1.3.1 Proteomic studies of HCMV infection 41 1.3.2 Affinity-purification mass spectrometry 44 1.4 Project aims 45 2 | Materials and Methods 48 2.1 Solutions 48 2.2. Cell Culture 51 2.2.1 Established cell lines 51 2.2.2 Cell culture conditions 51 2.2.3 Cell line passage 51 2.2.4 Cell counting 52 2.2.5 Stable cell line generation 52 2.2.6 Cryopreservation 53 2.2.7 Transient transfection 53 1 2.3 HCMV 54 2.3.1 Propagation of HCMV strains 54 2.3.2 Purification of viral stocks 55 2.3.3 Viral titration 56 2.3.4 Infection for assays 56 2.3.5 Viral growth curve 57 2.4. Molecular Biology 57 2.4.1 Polymerase chain reaction 57 2.4.2 Agarose gel electrophoresis 58 2.4.3 DNA purification from an agarose gel 58 2.4.4 Gateway cloning 59 2.4.5 Bacterial transformation 61 2.4.6 Small-scale plasmid DNA preparation 62 2.4.7 Large-scale plasmid DNA preparation 63 2.4.8 Nucleic acid sample quantification 63 2.4.9 Glycerol stock generation 64 2.5 Construction of the expression vector library 64 2.5.1 Genes cloned from recombinant adenoviral vector library 64 2.5.2 Genes cloned from HCMV Merlin BAC or cDNA template 66 2.5.3 Synthesized genes 67 2.5.4 pHAGE-pSFFV vector control 68 2.5.5 Cloning of human genes 68 2.5.6 Site-directed mutagenesis 71 2.5.7 Construct sequencing 72 2.6. RT-QPCR 73 2.6.1 Cellular RNA Extraction 73 2.6.2 DNase treatment 73 2.6.3 Reverse-transcription reaction 74 2.6.4. qPCR 74 2.7. Immunoblotting 76 2.7.1 Preparation of cell lysates 76 2.7.2 Estimation of protein concentration 76 2.7.3 Immunoblotting 77 2.8 Flow Cytometry 79 2.9. Immunoprecipitation 80 2.9.1 Affinity-purification mass spectrometry 80 2.9.1.1 Viral infection 80 2.9.1.2 Cell lysis 80 2.9.1.3 Anti-V5 Immunoprecipitation 81 2.9.1.4 Protein precipitation 82 2.9.1.5 Trypsin digest 82 2.9.1.6 StageTip 82 2.9.1.7 LC-MS/MS 83 2.9.2 Immunoprecipitation for immunoblotting 84 2.10 Proteomic analysis of whole-cell lysates 85 2.10.1 Viral infection 85 2.10.2 Cell lysis 86 2.10.3 Reduction and alkylation of disulphide bonds 86 2.10.4 Protein digestion with LysC and Trypsin 86 2 2.10.5 Protein isolation with SepPak 87 2.10.6 Peptide labelling with Tandem Mass Tags 87 2.10.7 Offline high pH reversed phase fractionation 88 2.10.8 LC-MS3 88 2.11 Data analysis 89 2.11.1 Database and search parameters for protein identification 89 2.11.2 Interactor identification with CompPASS 91 2.11.3 Interaction database comparisons 96 2.11.4 Functional enrichment analysis 96 2.11.5 Domain association analysis 96 2.11.6 ORFL147C DNA and amino acid sequence alignment analysis 97 3 | Generating resources for AP-MS 99 3.1 Generation of the expression construct library 99 3.2 Detecting the expression of recombinant constructs 102 3.3 Generation of HCMV stock 110 3.4 Infection conditions 111 3.5 Discussion 113 4 | Optimising the immunoprecipitation protocol 117 4.1 Anti-V5 agarose beads 118 4.2 Bead volume per sample 120 4.3 Input material per immunoprecipitation reaction 122 4.4 Peptide and protein yields of V5 elutions 125 4.5 Peptide solubilising agents 126 4.6 Discussion 127 5 | HCMV-host protein interactions 131 5.1 Controls and correlation